There are two paralogs of SGK1, SGK2 and SGK3/CISK, which share 80% amino acid identity with SGK1 and with each other in their catalytic domains (Kobayashi et al., 1999; Liu et al., 2000; Dai et al., 1999).

PX domains were originally found as conserved domains in the p40phox and p47phox subunits of the neutrophil NADPH oxidase (phox) superoxide-generating complex (Ponting CP., (1996) Protein Sci). These domains are part of many proteins involved in intracellular protein trafficking, such as the sorting nexins. PX domains are phosphoinositide-binding domains that appear to be important for localization of these proteins to membranes (especially endosomes) enriched in phosphoinositides. In this respect, these domains resemble other domains such as the pleckstrin homology (PH), FYFE, FERM and ENTH domains. PKB/Akt contains a PH domain in its N terminus, which is important for PKB/Akt activation by phosphoinositide-3 kinases (PI-3Ks). This domain enables the colocalization with the 3- phosphoinositide-dependent protein kinase-1 (PDK1), which is known to phosphorylate and activate PKB/Akt. Similarly, SGK3’s PX domain is involved in SGK3 localization and activity: It is necessary for phosphoinositide binding, endosomal localization, and proper kinase activity. Moreover, structural studies indicate that it may play a role in dimerization of the kinase. With respect to their physiological role(s), it has been shown in vitro that both the SGK2 and SGK3 enzymes have the same phosphorylation consensus as SGK1 (and PKB/Akt), namely R-X-R-X-X-(S/T). It is likely, however, that other factors, such as surrounding amino acids, subcellular localization, or cofactors are important for the specificity of and functional differences between the enzymes. For example, in Xenopus A6 cells, only SGK1 and not the coexpressed PKB modulates the activity of the epithelial Na+ channel (ENaC) (Arteaga MF. et al., (2005) Am J Physiol Renal Physiol).

The role of SGK2 has mainly been studied in heterologous expression systems such as Xenopus laevis oocytes or HEK293 cells and with respect to numerous transport and channel proteins. These studies revealed that SGK2 can stimulate the activity of K+ channels such as the voltage- gated K+ channel Kv1.3 (Gamper et al., (2002) Pflügers Arc,; Henke et al., (2004) J Cell Physiol), Na+-K+-ATPase (Henke G. et al., (2002) Kidney Blood Press Res), KCNE1 (Embark et al., (2003) Pflügers Arch), ENaC (Friedrich et al., (2003) Pflügers Arch), the glutamate transporter EEAT4 (Böhmer et al., (2004) Biochem Biophys Res Commun), and the glutamate receptors GluR6 (Strutz-Seebohm et al., (2005) J Physiol) and GluR1 (Strutz-Seebohm et al., (2005a) J Physiol). All of these transport proteins are also stimulated in the same cellular systems by SGK1, SGK3, and/or PKB; hence, the physiological relevance of these findings has to be considered with caution. To define more precisely the role of SGK2, it will be necessary to carry out additional studies, using more relevant cell or animal systems and knocking down SGK2 by either RNA interference protocols or by gene inactivation. SGK3/CISK, which is better characterized than SGK2, was identified in a screen for antiapoptotic genes (Liu et al., (2000) Curr Biol) and found to act downstream of the PI-3K pathway and in parallel with PKB/Akt. Moreover, it was demonstrated to phosphorylate and inhibit Bad (a proapoptotic protein) and FKHRL1, a proapoptotic transcription factor. Knockout (ko) mice have been generated; these mice are viable and fertile and have normal Na+ handling and glucose tolerance, as opposed to the KO mice of SGK1 or PKB/Akt2 (McCormick et al., (2004) Mol Biol Cell; Garofalo et al., (2003) J Clin Invest; Wulff et al., (2002) J Clin Invest). However, they display after birth a defect in hair follicle development, a defect preceded by disturbances in the β-catenin/Lef1 gene regulation (McCormick et al., (2004) Mol Biol Cell).

The three enzymes differ in the region N-terminal of the C-terminal catalytic domain: SGK2 contains a relatively short N terminus (98 amino

[page 11]

acids), with no discernable domain, whereas SGK3 has a longer N terminus (162 amino acids) comprising a phox homology (PX) domain (Xu et al., 2001). PX domains were originally found as conserved domains in the p40phox and p47phox subunits of the neutrophil NADPH oxidase (phox) superoxide-generating complex (Ponting 1996). These domains are part of many proteins involved in intracellular protein trafficking, such as the sorting nexins (Worby and Dixon 2002). PX domains are phosphoinositide-binding domains that appear to be important for localization of these proteins to membranes (especially endosomes) enriched in phosphoinositides. In this respect, these domains resemble other domains such as the pleckstrin homology (PH), FYFE, FERM and ENTH domains. PKB/Akt contains a PH domain in its N terminus, which is important for PKB/Akt activation by phosphoinositide-3 kinases (PI-3Ks). This domain enables the colocalization with the 3-phosphoinositide-dependent protein kinase-1 (PDK-1), which is known to phosphorylate and activate PKB/Akt. Similarly, SGK3’s PX domain is involved in SGK3 localization and activity: It is necessary for phosphoinositide binding, endosomal localization, and proper kinase activity (Xu et al., 2001; Liu et al., 2000). Moreover, structural studies indicate that it may play a role in dimerization of the kinase (Xing et al., 2004). With respect to their physiological role(s), it has been shown in vitro that both the SGK2 and SGK3 enzymes have the same phosphorylation consensus as SGK1 (and PKB/Akt), namely R-X-R-X-X-(S/T) (Kobayashi et al., 1999). It is likely, however, that other factors, such as surrounding amino acids, subcellular localization, or cofactors are important for the specificity of and functional differences between the enzymes. For example, in Xenopus A6 cells, only SGK1 and not the coexpressed PKB modulates the activity of the epithelial Na+ channel (ENaC) (Artega et al., 2005). The role of SGK2 has mainly been studied in heterologous expression systems such as Xenopus laevis oocytes or HEK293 cells and with respect to numerous transport and channel proteins. These studies revealed that SGK2 can stimulate the activity of K+ channels such as the voltage- gated K+ channel Kv1.3 (Gamper et al., 2002; Henke et al., 2004), Na+,K+-ATPase (Henke et al., 2002), KCNE1 (Embark et al., 2003), ENaC (Friedrich et al., 2003), the glutamate transporter EEAT4 (Bohmer et al., 2004), and the glutamate receptors GluR6 (Strutz-Seebohm et al., 2005) and GluR1 (Strutz-Seebohm et al., 2005a). All of these transport proteins are also stimulated in the same cellular systems by SGK1, SGK3, and/or PKB; hence, the physiological relevance of these findings has to be considered with caution. To define more precisely the role of SGK2, it will be necessary to

[page 12]

carry out additional studies, using more relevant cell or animal systems and knocking down SGK2 by either RNA interference protocols or by gene inactivation. SGK3/CISK, which is better characterized than SGK2, was identified in a screen for antiapoptotic genes (Liu et al., 2000) and found to act downstream of the PI-3K pathway and in parallel with PKB/Akt. Moreover, it was demonstrated to phosphorylate and inhibit Bad (a proapoptotic protein) and FKHRL1, a proapoptotic transcription factor. Knockout (KO) mice have been generated; these mice are viable and fertile and have normal Na+ handling and glucose tolerance, as opposed to the KO mice of SGK1 or PKB/Akt2 (McCormick et al., 2004; Garofalo et al., 2003; Wulff et al., 2002; Cho et al., 2001). However, they display after birth a defect in hair follicle development, a defect preceded by disturbances in the β-catenein/Lef1 gene regulation (McCormick et al., 2004).

SGK1 mRNA expression is established very early in embryonic development, as indicated by in situ hybridizations on whole-mount preparations of mouse embryo (Lee et al., (2001) Mech Dev). By embryonic day (E) 8.5, SGK1 is already highly expressed in the decidua and yolk sac. By days E9.5– E12.5 it is found in the developing heart, eye, and lung, and it becomes highly expressed by days E13.5–E16.5 in the brain choroid plexus, kidney distal tubules, bronchi/bronchiole, adrenal glands, liver, thymus, and intestine (Lee et al., (2001) Mech Dev). In contrast to SGK1 and SGK3, SGK2 reveals a more restricted distribution and is highly abundant only in the liver, kidney, and pancreas, where it is found in two different SGK2 species, referred to as SGK2α and SGK2β (Kobayashi et al., (1999) Biochem J). SGK isoform expression varies also between cell lines cultured in vitro. Similar to its expression pattern in vivo, SGK1 is broadly expressed in cultured cells and is readily detectable in, for example, hepatoma cells, fibroblasts and mammary tumor cells (Webster et al., (1993) J Biol Chem; Kobayashi et al., (1999) Biochem J). By contrast, SGK2 mRNA is expressed in hepatoma cells but not in fibroblasts, whereas SGK3 is found in fibroblasts but not in hepatoma cells. Remarkably, all three SGK isoforms are expressed in cells derived from the renal cortical collecting duct (Naray-Fejes-Toth et al., (2004) Proc Natl Acad Sci USA).

SGK2β (Kobayashi et al., 1999). SGK isoform expression varies also between cell lines cultured in vitro. Similar to its expression pattern in vivo, SGK1 is broadly expressed in cultured cells and is readily detectable in, for example, hepatoma cells, fibroblasts, and mammary tumor cells (Webster et al., 1993; Kobayashi et al., 1999). By contrast, SGK2 mRNA is expressed in hepatoma cells but not in fibroblasts, whereas SGK3 is found in fibroblasts but not in hepatoma cells. Remarkably, all three SGK isoforms are expressed in cells derived from the renal cortical collecting duct (Naray-Fejes-Toth et al., 2004).

Although SGK isoforms are expressed in various tissues and cell types, the role of SGK1 in aldosterone-dependent regulation of Na+ homeostasis is the best-studied function of these kinases with respect to epithelial ion transport. The kidneys play a pivotal role in the maintenance of Na+ homeostasis. Urinary Na+ excretion must be tightly regulated to maintain a constant extracellular volume during varying dietary Na+ intake and extrarenal Na+ losses. The final control of renal Na+ excretion is achieved by the ASDN i.e. the late distal convoluted tubule, the connecting tubule and the cortical as well as the medullary collecting ducts (CCD and MCD respectively) (Loffing J. et al., (2001) Am J Physiol Renal Physiol).

1.5 Role of SGK1 in Aldosterone Dependent Na+ Reabsorption

Although SGK isoforms are expressed in various tissues and cell types, the role of SGK1 in aldosterone-dependent regulation of Na+ homeostasis is the best-studied function of these kinases with respect to epithelial ion transport. The kidneys play a pivotal role in the maintenance of Na+ homeostasis. Urinary Na+ excretion must be tightly regulated to maintain a constant extracellular volume during varying dietary Na+ intake and extrarenal Na+ losses. The final control of renal Na+ excretion is achieved by the ASDN i.e. the late distal convoluted tubule, the connecting tubule, and the cortical as well as the medullary collecting ducts (CCD and MCD respectively) (Loffing et al., 2001).

Transepithelial Na+ transport in these segments is accomplished by Na+ entry into the epithelial cells via the epithelial Na+ channel (ENaC) in the luminal membrane and by exit of Na+ through the Na+, K+-ATPase in the basolateral plasma membrane. ENaC represents the rate limiting step in this process and is highly regulated (Kellenberger and Schild 2002). It is composed of three subunits (α, β and γ) (Canessa et al., 1994; Canessa et al., 1994; Lingueglia et al., 1993; Lingueglia et al., 1994) with a stoichiometry of 2α1β1γ (Firsov et al., 1998), although other stoichiometries have also been proposed (octa- or nonamers) (Eskandari et al., 1999; Snyder et al., 1998). Its subunits have a similar topology, with two transmembrane domains, one extracellular loop, and two cytoplasmic ends (Renard et al., 1994; Canessa et al., 1994; Snyder et al., 1994). Each subunit also contains, at its C-terminal end, a PY-motif (P-P-X-Y, where P is a proline, Y a tyrosine, and X any amino acid), which is known as protein:protein interaction motifs that can interact with tryptophan (W)-rich WW domains (Chen and Sudol 1995; Staub and Rotin 1996). The importance of these PY-motifs for ENaC regulation has been recognized by the findings that most cases of Liddle’s syndrome [(Liddle et al., 1963)]

Note that the first sentence of the second paragraph makes little sense, as it is a combination of two unrelated half-sentences of the source, the first one ending just at the page break from page 15 to page 16.

The stimulatory effect of SGK1 on ENaC is related both to an increased number of channels in the plasma membrane (Lang F. et al., (2000) Proc Natl Acad Sci USA; Loffing J. et al., (2001) Am J Physiol Renal Physiol; Alvarez de la Rosa D. et al., (1999) J Biol Chem) and an activation of channels already present in the membrane (Diakov A. and Korbmacher C., (2004) J Biol Chem). The first effect likely involves the action of Nedd4-2, as there are several consensus phosphorylation motifs (2–3 depending on the splice variant) on Nedd4-2 and a PY-motif on SGK1 that may serve as a binding site for Nedd4-2.

The stimulatory effect of SGK1 on ENaC is related both to an increased number of channels in the plasma membrane (Lang et al., 2000; Loffing et al., 2001; Alvarez de la Rosa et al., 1999) and an activation of channels already present in the membrane (Diakov and Korbmacher 2004). The first effect likely involves the action of Nedd4-2, as there are several consensus phosphorylation motifs (2–3 depending on the splice variant) on Nedd4-2 and a PY-motif on SGK1 that may serve as a binding site for Nedd4-2. In Xenopus oocytes, SGK1 induces Nedd4-2 phosphorylation on two of these phosphorylation sites (primarily Ser444, but also Ser338) (Embark et al., 2004; Palmada et al., 2004; Debonneville et al., 2001), which decreases the interaction of Nedd4-2 with ENaC and finally leads to an enhanced expression and activity of ENaC at the cell surface (Debonneville et al., 2001).

This inhibitory effect of SGK1 on Nedd4-2 likely involves 14-3-3 proteins as phosphorylation of Ser444 in Nedd4-2 creates a possible binding site for such proteins, an inherited form of salt-sensitive hypertension are caused by mutations in the genes encoding β- and γ -ENaC (Hansson JH. et al., (1995) Proc Natl Acad Sci USA; Shimkets RA. et al., (1994) Cell). These mutations invariably cause either the deletion or the mutation of the PY-motifs on these subunits. When such Liddle channels are expressed in heterologous systems, increases in both the density at the cell surface and the open probability of ENaC are observed (Firsov et al., (1996) Proc Natl Acad Sci USA; Snyder PM. et al., (1994) Cell; Schild et al., (1995) Proc Natl Acad Sci USA; Schild et al., (1996) EMBO J). Loffing and his coworkers, has demonstrated that these PY-motifs are the binding sites for ubiquitin-protein ligases of the Nedd4/Nedd4-like family (Kamynina E. et al., (2001) EMBO J) and particularly of Nedd4-2 (Kamynina E. et al., (2001) EMBO J; Snyder PM. et al., (2004) J Biol Chem).

It is thought that Nedd4-2 binds via its WW domains with the PY-motifs of ENaC and ubiquitylates ENaC on its own α and γ subunits, consequently leading to the internalization and degradation of ENaC in the endosomal/lysosomal system (Snyder PM. et al., (2004) J Biol Chem). In Liddle’s syndrome, this mechanism is impaired owing to the inactivation of a PY-motif, causing the accumulation of ENaC at the plasma membrane (Kamynina E. and Staub O., (2002) Am J Physiol Renal Physiol). The activity of ENaC and the Na+, K+-ATPase is tightly regulated by aldosterone and by SGK1 (Vallon V. et al., (2005) Am J Physiol Regul Integr Comp Physiol; Bhargava A. et al., (2004) Trends Endocrinol Metab).

[...], an inherited form of salt-sensitive hypertension are caused by mutations in the genes encoding β- and γ -ENaC (Hansson et al., 1995; Shimkets et al., 1994). These mutations invariably cause either the deletion or the mutation of the PY-motifs on these subunits. When such Liddle channels are expressed in heterologous systems, increases in both the density at the cell surface and the open probability of ENaC are observed (Firsov et al., 1996; Snyder et al., 1995; Schild et al., 1995; Schild et al., 1996). Loffing and his coworkers, has demonstrated that these PY-motifs are the binding sites for ubiquitin-protein ligases of the Nedd4/Nedd4-like family (Kamynina et al., 2001; Kamynina et al., 2001a ) and particularly of Nedd4-2 (Kamynina et al., 2001; Kamynina et al., 2001a; Snyder et al., 2004). It is thought that Nedd4-2 binds via its WW domains with the PY-motifs of ENaC and ubiquitylates ENaC on its α and γ subunits, consequently leading to the internalization and degradation of ENaC in the endosomal/lysosomal system (Snyder et al., 2004). In Liddle’s syndrome, this mechanism is impaired owing to the inactivation of a PY-motif, causing the accumulation of ENaC at the plasma membrane (Kamynina and Staub 2002). The activity of ENaC and the Na+, K+-ATPase is tightly regulated by aldosterone and by SGK1 (Kellenberger and Schild 2002; Vallon et al., 2005; Bhargava et al., 2004). [...] This inhibitory effect of SGK1 on Nedd4-2 likely involves 14-3-3 proteins as phosphorylation of Ser444 in Nedd4-2 creates a possible binding site for such proteins

Anmerkungen

The source is not given.

Note that the first sentence makes little sense, as it is a combination of two unrelated half-sentences of the source. Apparently the last line of the source on page 16 has not been continued with the first line on page 17, but with the first line on page 16 again, leading to this result. Also, the source writes Ser444, Dsa writes Ser444 constistently.

Experiments in native Xenopus A6 cells expressing endogenous SGK1 and ENaC further confirmed that the action of SGK1 on ENaC is complex and likely involves (a) increases in the subunit abundance in the plasma membrane and (b) activation of channels already in the plasma membrane combined with an increase in ENaC open probability (Alvarez de la Rosa D. et al., (2004) J Physiol). However, in this model the stimulatory effect on ENaC channel activity cannot be explained by a direct SGK1-dependent phosphorylation of α-ENaC because Xenopus α-ENaC does not contain the SGK1 consensus phosphorylation motif. That direct phosphorylation of ENaC at the SGK1 consensus site is not essential for ENaC activation is also supported by data from Lang and coworkers (Lang F. et al., (2000) Proc Natl Acad Sci USA; Friedrich B. et al., (2002) Kidney Blood Press Res) that showed that channels with a serine-to-alanine mutation within the consensus site of α-ENaC are still rigorously upregulated by coexpression of SGK1 in Xenopus oocytes.

In addition to this indirect action of SGK1 on ENaC cell surface abundance, it was proposed that SGK1 can directly interact with ENaC (Wang et al., 2001) and increase ENaC channel activity by phosphorylating the α-ENaC subunits (Diakov and Korbmacher 2004). Diakov & Korbmacher (2004) used outside-out membrane patches of X. laevis oocytes expressing rat ENaC to demonstrate that addition of recombinant, constitutively active SGK1 directly stimulates ENaC currents two- to threefold. An alanine mutation of the serine residue in the SGK1 consensus R-X-R-X-X-S phosphorylation motif abolishes the stimulatory effect on ENaC in this experimental setting. Experiments in native Xenopus A6 cells expressing endogenous SGK1 and ENaC further confirmed that the action of SGK1 on ENaC is complex and likely involves (a) increases in the subunit abundance in the plasma membrane and (b) activation of channels already in the plasma membrane combined with an increase in ENaC open probability (Alvarez de la Rosa et al., 2004). However, in this model the stimulatory effect on ENaC channel activity cannot be explained by a direct SGK1-dependent phosphorylation of α-ENaC because Xenopus α-ENaC does not contain the SGK1 consensus phosphorylation motif. That direct phosphorylation of ENaC at the SGK1 consensus site is not essential for ENaC activation is also supported by data from Lang and coworkers (Lang et al., 2000; Friedrich et al., 2002) that showed that channels with a serine-to-alanine mutation within the consensus site of α-ENaC are still rigorously upregulated by coexpression of SGK1 in Xenopus oocytes. NDRG-2, which is an aldosterone-induced protein in the ASDN, is another target of SGK1 (Boulkroum et al., 2002; Murray et al., 2004). Although the functional role of NDRG-2 in the ASDN is not known, this protein may also have some function in the SGK1-dependent signaling cascade related to Na+ transport. As an aldosterone-induced protein, SGK1 is thought to mediate at least some of the physiological effects of aldosterone on ENaC and Na+,K+-ATPase. The stimulatory effect of aldosterone (or of dexamethasone) on SGK1 expression has now been firmly documented in several studies on various in vitro and in vivo systems, including Xenopus A6 cells (Bhargava et al., 20004), primary rabbit CCD cells (Narey-Fejes-Toth et

At least part of the stimulatory effect of aldosterone on SGK1 appears to be mediated by activation of the MR, as indicated by findings in primary rabbit collecting duct cells in vitro (Narey-Fejes-Toth et al., (1999) [sic] J Biol Chem) and kidneys in vivo (Bhargava A. et al., (2001) Endocrinology). Consistently, physiologically relevant concentrations of aldosterone are sufficient to significantly induce SGK1 mRNA in the renal cortex and outer medulla (Muller OG. et al., (2003) J Am Soc Nephrol). The physiological importance of aldosterone in SGK induction is also supported by the fact that dietary Na+ restriction, which physiologically increases plasma aldosterone, induces SGK1 mRNA in the renal cortex (Farjah M. et al., (2003) Hypertension). The aldosterone-dependent induction of SGK1 occurs specifically in the ENaC-positive cells of the ASDN, whereas SGK1 expression in other nephron portions such as the thick ascending limb or the proximal tubule is not increased by aldosterone. Likewise, the high level of expression of SGK1 in the renal papilla is not further stimulated by aldosterone, suggesting that SGK1 expression at this site is controlled by factors other than aldosterone. The renal papilla plays an important role for the urinary concentration mechanism, and the cells in the renal papilla can be exposed to a large variation in extracellular osmolarity depending on the requirements for diuresis to antidiuresis. SGK1 expression is strongly modulated by osmotic cell shrinkage and swelling (Waldegger S. et al., (1997) Proc Natl Acad Sci USA; Rozansky DJ. et al., (2002) Am J Renal Physiol), and it is therefore conceivable that SGK1 participates in the functional adaptation of the renal papilla cells to fluctuation of extracellular osmolarity.

At least part of the stimulatory effect of aldosterone on SGK1 appears to be mediated by activation of the MR, as indicated by findings in primary rabbit collecting duct cells in vitro (Naray-Fejes-Toth et al., 1999) and kidneys in vivo (Bhargava et al., 2001). Consistently, physiologically relevant concentrations of aldosterone are sufficient to significantly induce SGK1 mRNA in the renal cortex and outer medulla (Muller et al., 2003). The physiological importance of aldosterone in SGK induction is also supported by the fact that dietary Na+ restriction, which physiologically increases plasma aldosterone, induces SGK1 mRNA in the renal cortex (Farjah et al., 2003). The aldosterone-dependent induction of SGK1 occurs specifically in the ENaC-positive cells of the ASDN, whereas SGK1 expression in other nephron portions such as the thick ascending limb or the proximal tubule is not increased by aldosterone. Likewise, the high level of expression of SGK1 in the renal papilla is not further stimulated by aldosterone, suggesting that SGK1 expression at this site is controlled by factors other than aldosterone. The renal papilla plays an important role for the urinary concentration mechanism, and the cells in the renal papilla can be exposed to a large variation in extracellular osmolarity depending on the requirements for diuresis to antidiuresis. SGK1 expression is strongly modulated by osmotic cell shrinkage and swelling (Waldegger et al., 1997; Rozansky et al., 2002), and it is therefore conceivable that SGK1 participates in the functional adaptation of the renal papilla cells to fluctuation of extracellular osmolarity. Consistent with this notion, recent data suggest that SGK1 mediates the osmotic induction of the type A natriuretic peptide receptor (NPR-A) in rat inner MCD cells (Chen et al., 2004). Aldosterone also controls SGK1 expression in the distal colon (Coric et al., 2004; Bhargava et al., 2001). Aldosterone-dependent Na+ reabsorption at this site may help to limit extrarenal Na+ losses during conditions of dietary Na+ restriction. Transepithelial Na+ transport is achieved mainly by epithelial cells that are situated at the tips of colonic crypts and that express high levels of

[page 19]

ENaC (Coric et al., 2004) and Sgk1 (Waldegger et al., 1999; Coric et al., 2004). In spite of these data pointing to aldosterone-dependent regulation of ENaC via SGK1, recent Western blot and immunohistochemical studies on rat kidney and colon, which reported no or rather modest aldosterone-dependent induction of SGK1 at the protein level, were interpreted to question the significance of aldosterone-dependent induction of SGK1 for ENaC-mediated Na+ transport regulation (Coric et al., 2004). Support for a functional significance of SGK1 in regulation of transepithelial Na+ transport comes from experiments in X. laevis A6 cells and in mouse M1 CCD cells. Transfection of A6 or M1 cells with SGK1 leads to an increase in transepithelial Na+ transport, whereas transfection of a dominant-negative “kinase-dead” SGK1 mutant or an antisense SGK1 transcript abolishes dexamethasone- and/or insulin-dependent regulation of transepithelial Na+ transport (Alvarez de Rosa et al., 2003; Faletti et al., 2002). Likewise, the use of interfering RNA to knockdown SGK1 expression in A6 cells results in a significant reduction in SGK1 protein levels and a ~50% reduction in dexamethasone-induced short-circuit currents (Bhargava et al., 2004). Consistent with these in vitro findings, experiments in SGK1 KO (sgk1-/-) mice supported the importance of SGK1 for aldosterone-dependent regulation of renal Na+ transport (Wulff et al., 2002).

Under dietary Na+ restriction, activated compensatory mechanisms are no longer sufficient to keep the mice in Na+ balance, and mice disclosed significant loss in renal NaCl and in body weight. Experiments on collecting ducts perfused ex vivo revealed significantly lower transepithelial amiloride-sensitive potential differences, consistent with a reduced Na transport activity in the CCD. Although apical localization of ENaC was seen in both Na+- restricted sgk1+/+ and sgk1−/− mice, the apical localization of ENaC is inappropriately low in the sgk1−/− mice given the several fold higher plasma aldosterone levels in the KO mice.

Nevertheless, these data, together with the rather mild phenotype of sgk1−/− mice, as compared to the much more severe and life-threatening phenotypes of MR or ENaC ko mice, suggest that (a) aldosterone-dependent control of ENaC function does not solely rely on the induction and activation of SGK1 and (b) some redundancy exists in the signal transduction pathway that controls ENaC activity. Consistent with these ideas, Loffing and his coworkers found significant phosphorylation of the SGK1 target Nedd4-2 in mouse mpkCCDcl4 cells in vitro and in rat collecting ducts in vivo in the absence of any aldosterone and detectable SGK1 protein expression (Flores SY. et al., (2005) J Am Soc Nephrol). In addition to aldosterone-dependent regulation of renal Nareabsorption [sic] , SGK1 appears to be involved also in the regulation of aldosterone-induced salt appetite. Sgk1+/+ and sgk1−/− mice show a similar salt intake under standard conditions. Treatment with the synthetic aldosterone analogue deoxycorticosterone-acetate (DOCA) increases Na+ intake much more in sgk1+/+ mice than in sgk1−/− mice. The underlying mechanism for the reduced mineralocorticoid-induced salt intake is unclear (Vallon V. et al., (2005) Am J Physiol Regul Integr Comp Physiol).

b) Role of SGK1 in renal K+ secretion

Aside from its stimulatory effect on renal Na+ reabsorption, aldosterone has strong kaliuretic action. Renal K+ secretion also takes place in the ASDN and is likely mediated by the renal outer medullary K+ channel ROMK. ROMK is coexpressed with ENaC in the ASDN cells, and Na+ reabsorption via ENaC provides the necessary driving force for K+ secretion. Consistently, pharmacological inhibition (i.e., by amiloride) or genetic loss of function (i.e., pseudohypoaldosteronism (PHA) type 1) of ENaC lower renal K+ secretion and predispose one to hyperkalemia.

It remains unresolved whether the kaliuretic effect of aldosterone is entirely secondary to the activation of ENaC-mediated Na+ reabsorption or whether aldosterone directly regulates ROMK function.

Under a standard diet, the KO mice have unaltered Na+ excretion as compared to their wildtype littermates. However, plasma aldosterone levels are significantly increased in sgk1-/- mice, suggesting extracellular volume contraction. Under dietary Na+ restriction, activated compensatory mechanisms are no longer sufficient to keep the mice in Na+ balance, and mice disclosed significant loss in renal NaCl and in body weight. Experiments on collecting ducts perfused ex vivo revealed significantly lower transepithelial amiloride-sensitive potential differences, consistent with a reduced Na+ transport activity in the CCD. Although apical localization of ENaC was seen in both Na+-restricted sgk+/+ [sic] and sgk1-/- mice, the apical localization of ENaC is inappropriately low in the sgk1-/- mice given the severalfold higher plasma aldosterone levels in the KO mice. Nevertheless, these data, together with the rather mild phenotype of sgk1-/- mice, as compared to the much more severe and life-threatening phenotypes of MR or ENaC KO mice, suggest that (a) aldosterone-dependent control of ENaC function does not solely rely on the induction and activation of SGK1 and (b) some redundancy exists in the signal transduction pathway that controls ENaC activity. Consistent with these ideas, Loffing and his coworkers found significant phosphorylation of the SGK1 target Nedd4-2 in mouse mpkCCDcl4 cells in vitro

[page 20]

and in rat collecting ducts in vivo in the absence of any aldosterone and detectable SGK1 protein expression (Flores et al., 2005). In addition to aldosterone-dependent regulation of renal Na+ reabsorption, SGK1 appears to be involved also in the regulation of aldosterone-induced salt appetite. Sgk1+/+ and sgk1-/- mice show a similar salt intake under standard conditions. Treatment with the synthetic aldosterone analogue deoxycorticosterone-acetate (DOCA) increases Na+ intake much more in Sgk1+/+ mice than in sgk1-/- mice. The underlying mechanism for the reduced mineralocorticoid-induced salt intake is unclear (Vallon et al., 2005).

1.6 Role of SGK1 in Renal K+ Secretion

Aside from its stimulatory effect on renal Na+ reabsorption, aldosterone has strong kaliuretic action. Renal K+ secretion also takes place in the ASDN and is likely mediated by the renal outer medullary K+ channel ROMK. ROMK is coexpressed with ENaC in the ASDN cells, and Na+ reabsorption via ENaC provides the necessary driving force for K+ secretion. Consistently, pharmacological inhibition (i.e., by amiloride) or genetic loss of function [i.e., pseudohypoaldosteronism (PHA) type 1] of ENaC lower renal K+ secretion and predispose one to hyperkalemia. It remains unresolved whether the kaliuretic effect of aldosterone is entirely secondary to the activation of ENaC-mediated Na+ reabsorption or whether aldosterone directly regulates ROMK function.

The in vivo significance of SGK1 in regulation of renal K+ transport was recently analyzed in SGK1 KO mice. These mice indeed show a disturbed adaptation to an acute and chronic K+ load, but, as indicated by electrophysiological and immunohistochemical data obtained from these mice after a chronic potassium load, this maladaptation likely is related to altered ENaC (or Na+, K+-ATPase) activity in the ASDN cells rather than to inhibition of ROMK cell surface targeting or activity (Huang DY. et al., (2004) J Am Soc Neprhol).

The in vivo significance of SGK1 in regulation of renal K+ transport was recently analyzed in SGK1 KO mice. These mice indeed show a disturbed adaptation to an acute and chronic K+ load, but, as indicated by electrophysiological and immunohistochemical data obtained from these mice after a chronic potassium load, this maladaptation likely is related to altered ENaC (or Na+,K+-ATPase) activity in the ASDN cells rather than to inhibition of ROMK cell surface targeting or activity (Huang et al., 2004).

Mice deficient in SGK1 (sgk1–/–) were generated and bred as previously described (Huang et al., (2004) J Am Soc Nephol; Wulff et al., (2002) J Clin Invest; Huang et al., (2004) J Am Soc Nephol). In brief, a conditional targeting vector was generated from a 7-kb fragment encompassing the entire transcribed region on 12 exons. The neomycin resistance cassette was flanked by two loxP sites and inserted into intron 11. Exons 4–11, which code for the sgk1 kinase domain, were “floxed” by inserting a third loxP site into intron 3. A clone with a recombination between the first and third loxP site (type I recombination) was injected into C57BL/6 blastocytes. Male chimeras were bred to C57BL/6 and 129/SvJ females. Heterozygous SGK1-deficient mice were backcrossed to 129/SvJ wild-type mice (Charles River, Sulzfeld, Germany) for ten generations and then intercrossed to generate homozygous SGK1 knockout mice (sgk1−/−) and their wild type littermates (sgk1+/+).

Mice deficient in SGK1 (sgk1-/-) were generated and bred as previously described (Wulff et al., 2002; Huang et al., 2004). In brief, a conditional targeting vector was generated from a 7-kb fragment encompassing the entire transcribed region on 12 exons. The neomycin resistance cassette was flanked by two loxP sites and inserted into intron 11. Exons 4–11, which code for the sgk1 kinase domain, were “floxed” by inserting a third loxP site into intron 3. A clone with a recombination between the first and third loxP site (type I recombination) was injected into C57BL/6 blastocytes. Male chimeras were bred to C57BL/6 and 129/SvJ females. Heterozygous SGK1-deficient mice were backcrossed to 129/SvJ wild-type mice (Charles River, Sulzfeld, Germany) for ten generations and then intercrossed to generate homozygous SGK1 knockout mice (sgk1-/-) and their wild type littermates (sgk1+/+).

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The source is not mentioned.

It is not surprising that the two dissertations have used mice that have been treated identically, and it is efficient to describe this with the same words, but this should have been made transparent.

The tail cuff method has the advantage to be noninvasive and can provide reproducible results of systolic blood pressure if those precautions are taken into account (Kurtz et al., (2005) Arterioscler Tromb Vasc Biol). Systolic arterial blood pressure was determined by the tail-cuff method (IITC, model 179, Woodland Hills, California, USA) before, 7 weeks and 12 weeks following the initiation of DOCA/high-salt treatment. As reviewed recently, (Meneton P et al., (2000) J Am Soc Neprhol), the tail cuff approach to determine arterial blood pressure requires certain precautions to reduce the stress of the animals, including appropriate training of the mice over multiple days and adequate prewarming to dilate the tail artery.

III. 3. Blood pressure: Systolic arterial blood pressure was determined by application of the tail-cuff method. As reviewed recently (Meneton et al., 2000), the tail cuff approach to determine arterial blood pressure requires certain precautions to reduce the stress of the animals, including appropriate training of the mice over multiple days, prewarming to an ambient temperature of 29°C, measurement in a quiet, semidarkend and clean environment, and performance of the measurements by one person and during a defined day time, when blood pressure is stable (between 1-3 PM). All these precautions were taken in the present study. The tail cuff method has the advantage to be noninvasive and can provide reproducible results of systolic blood pressure if those precautions are taken into account (Kurtz et al., 2005).

Along those lines enhanced SGK1 expression has been observed in the salt sensitive Dahl rat (Farjah., 2003). In addition, moderately enhanced blood pressure is observed in individuals carrying a variant of the SGK1 gene, affecting as many as 5% of unselected Caucasians (Busjahn et al., 2002). In the same individuals increased body mass index and a shortening of the QT interval (Busjahn et al., 2002; Busjahn and Luft 2003) have been observed. The increased body mass index may be partially due to enhanced stimulation of the intestinal glucose transporter SGLT1 (Dieter et al., 2004), the accelerated cardiac repolarization due to enhanced activation of the cardiac K+ channel KCNE1 (Busjahn et al., 2004; Embark et al., 2003). Thus, altered regulation of carriers and channels by SGK1 could account for the coincidence of obesity, hypertension and altered cardiac action potential (Lang et al., 2003). [...]